The analysis of biological samples to determine the identity and amounts of various proteins for diagnostic or research purposes is performed by a wide variety of separation techniques, many of which involve the use of dyes to enable the clinician to locate, identify, and quantitate the proteins. The dyes are detectable and readable by visual observation or are machine-readable. Included among the many types of dyes that are used for this purposes are those that emit colors in the visible range or the ultraviolet range to the eye without treatment or activation, and fluorescent dyes which emit upon excitation.
The dyes are most commonly applied to the proteins after separation has occurred and while the proteins are isolated into individual spots or bands in the gel. Applying the dyes in this manner and removing excess dye is a labor-intensive and time-consuming procedure that is susceptible to handling difficulties, operator error and various nonuniformities. Dyes that are insoluble or poorly soluble in water must first be dissolved in an organic solvent before being applied to the protein spots or bands in the gel. As an alternative, dyes can be applied to the proteins in the sample before the separation is performed. This also involves first dissolving the dyes in an organic solvent. One difficulty with these methods is that the ratio of dye to protein must be carefully controlled to avoid overstaining the proteins. Overstaining can cause nonuniformities among the various different proteins in the amount of dye that is coupled to each protein. Overstaining can also change the apparent mobilities of the proteins during electrophoresis, and this can occur to different degrees among different proteins, particularly when the proportion of dye to protein is nonuniform.
One of the areas in which these dyes are used is the electrophoresis of proteins under denaturing conditions. Denaturing electrophoresis, and particularly SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), is a highly effective and efficient means of analyzing proteins in a sample, since it identifies proteins independently of the potentially interfering influences of tertiary or quaternary shape or the complexity of their subunits. The concerns raised by the use of dyes apply with particular force to denaturing electrophoresis in view of its widespread use and effectiveness.